Cryopreservation of early-stage male Black Sea trout (Salmo trutta labrax) germ cells and comparison of the whole tissue and enzymatically isolated cells

The high plasticity, orthe ability todifferentiate into various cell types, and capacity tobecome both gametes ofearly-stage germ cells (eGCs) allows them to be used for the long-term storage and recovery ofgenetic information. Inthis study, species-specific protocols (isolation period, enzymatic separation solution, incubation period, appropriate cryoprotectant, and cryopreservation protocol) were established for the isolation and long-term preservation ofBlack Sea trout eGCs. Inaddition, the difference between the application ofcryopreservation tothe whole gonad tissue and tothe enzymatically separated cells has been shown interms ofviability and the number ofcells obtained. According tothe cell count made from the histological sections ofthe testicular tissue throughout the year, the period with the highest concentration ofeGCs inthe tissue isbetween May and June. Toidentify the optimal enzymatic dissociation solution, wesubjected testicular tissues todigestion using various combinations oftrypsin (T) and collagenase (C) (0.3–0.5% T and 0.1–0.3% C). Our findings revealed that the 0.3% C provided the highest yield ofviable cells (90.9%). Wecryopreserved the enzymatically dissociated cells (EDCs) using six different cryoprotectants (Dimethyl sulfoxide, Glycerol, and CryoSOfree™, inboth L-15 and phosphate-buffered saline – PBS mediums). Additionally, whole tissue exclusively with CryoSOfree™. After storing the samples at–152C for two years, the group ofEDCs cryopreserved in L15+Glycerol exhibited the highest viability at 93.3%. Meanwhile, the whole tissue group yielded the greatest number of cells per ml after thawing, with a count of 33 210 000 cells. When considering both the viability (91.11%) and the number of cells obtained (33 210 000 cells/ml), the whole tissue cryopreservation group outperformed all the other methods.